Joint Transcriptomic Analysis of the Effect of Iron Concentration on Piglet Liver and Functional Validation of Iron Regulatory Genes
Abstract
1. Introduction
2. Materials and Methods
2.1. Test Animals and Feeding Conditions
2.2. Feed Preparation
2.3. Sample Collection
2.4. Indicators and Methods
2.4.1. Growth Performance
2.4.2. Serum Biochemical Indicators
2.4.3. Determination of Tissue Iron Content
2.4.4. Liver Transcriptome Sequencing
2.4.5. WGCNA Screening for Iron Metabolism Core Genes
2.4.6. qPCR to Detect the Expression of Relevant Genes
2.4.7. Isolation of Piglet Liver Tissue Cells
2.4.8. Cellular Iron Deficiency
2.4.9. Cellular Iron Supplementation
2.4.10. Cellular RNA Extraction
2.5. Statistical Analysis
3. Results
3.1. Piglet Growth Performance
3.2. Piglet Serum Parameters
3.3. Piglet Visceral Iron Content
3.4. Liver Transcriptome
3.4.1. Sequencing Results and Quality Control
3.4.2. Differential Expression Gene Screening
3.4.3. Functional Enrichment of DEGs
3.4.4. Weighted Gene Co-Expression Network Analysis
3.4.5. Screening and Relative Expression of Core Genes
3.5. Iron Metabolism and Gene Expression in Hepatocytes: Chelation, Supplementation, and Transcriptional Control
3.5.1. Hepatocyte Iron Deficiency Treatment
3.5.2. Hepatocyte Iron Supplementation
3.5.3. Hepatocyte Gene Expression
4. Discussion
4.1. Layered Divergence Between In Vivo and In Vitro Iron Responses
4.2. Endocrine–Inflammatory Coupling Explains the In Vivo Upregulation of FGF21 and SAA Family
4.3. Metal-Responsive Transcription and ROS Feedback Underpin Non-Monotonic Patterns of FNDC1 and ETNPPL
4.4. Iron Trafficking and Cell-Type Heterogeneity Reconcile “Stable Total Iron” with Altered Signaling
4.5. Context Matters: Reconciling Apparent Inconsistencies with Prior Literature (e.g., TFR1, EPO)
4.6. Limitations and Next Steps: Hormone/Cytokine Complementation, Co-Culture, and Joint FPN/Ferritin/ROS Readouts
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Ingredient | Content (%) | Nutrient Level * | |
---|---|---|---|
Corn | 30.0 | CP (%) | 19.0 |
Soybean meal | 15.0 | Ca (%) | 0.80 |
Expanded soybean | 15.0 | Fe (mg/kg) | 76.8 |
Rice | 10.0 | Ap (%) | 0.42 |
Flour | 6.0 | Lys (%) | 1.22 |
Glucose | 4.0 | Met (%) | 0.39 |
Peruvian fish meal | 3.0 | Methionine + cystine (%) | 0.67 |
Citric acid | 1.50 | Thr (%) | 0.23 |
Vegetable oil | 1.00 | DE (MJ/kg) | 13.38 |
Fine stone powder | 0.84 | ||
Calcium hydrogen Phosphate | 0.81 | ||
Lysine | 0.18 | ||
Fungicide | 0.10 | ||
DL-methionine | 0.08 | ||
Premix | 0.49 |
Genes | Genes Accession Primer Sequence (5′–3′) | Amplification Length/bp |
---|---|---|
LOC106504547 | F:CCTGAAGGGCCAGATCAAGA R:TGACATTTTCTCTGCGTTCGG | 119 |
LOC100153899 | F:AGTCACCGACCCCAGTCAA R:GCTTTATTGCTGTGCGAGGTC | 204 |
ETNPPL | F:ATGCCAAATGAAGCACGCTC R:CGCTTGTGGCTGGGATTTTC | 74 |
SAA2 | F:ACCCCAACCACTTCAGACCT R:AGCAGTCCATCTCCTAAGCATTT | 166 |
SAA3 | F:GCTTTCCACGGGCATCATTT R:CATGTCCGAGTAGGCTCTCC | 121 |
FGF21 | F:GAAGCCCACCTGGAGATCAG R:GATCCGTACAGTCTCCCGTC | 167 |
RNF125 | F:CTCTGCTCTGCAGTTGAGGT R:CTTAGTTGAGGCGTGGAGGC | 104 |
ITIH4 | F:CATGAGGGGCAGGAAAATCCA R:GCCCTTCTCCTGGGTGCTTC | 120 |
FNDC1 | F:GGCATCCGAGTGGACAAAGA R:ACAATGGTTCGACCGTCTCC | 160 |
INHBE | F:GGCTACACTTGAGCAGTCGT R:GGACCGAGGAGTAGACAGGT | 197 |
Items 1 | A (0 mg/kg) | B (100 mg/kg) | C (200 mg/kg) | p-Value |
---|---|---|---|---|
IBW (kg) | 13.64 ± 0.54 | 13.47 ± 0.51 | 13.59 ± 0.70 | 0.97 |
FBW (kg) | 27.26 ± 1.04 | 27.05 ± 0.69 | 26.03 ± 1.15 | 0.65 |
ADG (g/day) | 425.44 ± 23.00 | 424.48 ± 14.49 | 388.8 ± 25.00 | 0.41 |
ADFI (g/day) | 921.19 ± 39.68 | 917.99 ± 25.83 | 860.56 ± 40.06 | 0.84 |
F/G | 2.19 ± 0.10 | 2.16 ± 0.06 | 2.20 ± 0.11 | 0.43 |
Items 1 | A (0 mg/kg) 2 | B (100 mg/kg) | C (200 mg/kg) | p-Value |
---|---|---|---|---|
EPO content (mIU/mL) | 4.49 ± 0.14a | 4.90 ± 0.22ab | 5.14 ± 0.17b | 0.058 |
HB content (μg/mL) | 66.76 ± 2.30 | 68.64 ± 1.12 | 64.68 ± 2.36 | 0.4 |
HIF-1 content (pg/mL) | 343.39 ± 3.15a | 374.22 ± 12.67b | 414.28 ± 8.94c | <0.0001 |
Serum iron level (mg/L) | 13.06 ± 0.76a | 10.73 ± 0.48b | 11.75 ± 0.76ab | 0.07 |
TFR content (nmol/L) | 16.27 ± 0.28a | 19.64 ± 1.12b | 19.54 ± 0.56b | 0.008 |
HEPC content (ng/mL) | 1182.83 ± 42.57 | 1208 ± 66.32 | 1227.33 ± 56.40 | 0.85 |
TIBC content (μHBmol/L) | 147.83 ± 8.72 | 149.36 ± 7.66 | 149.36 ± 6.41 | 0.26 |
GSH-PX viability (enzyme viability units) | 992.45 ± 118.91 | 904.15 ± 86.84 | 904.15 ± 99.84 | 0.66 |
MDA content (nmol/mL) | 1.75 ± 0.48 | 1.61 ± 0.12 | 1.61 ± 0.17 | 0.9 |
Items 1 | A (0 mg/kg) 2 | B (100 mg/kg) | C (200 mg/kg) | p-Value |
---|---|---|---|---|
spleen (mg/kg) | 198.78 ± 44.22 | 135.84 ± 16.42 | 180.70 ± 36.95 | 0.21 |
liver (mg/kg) | 79.91 ± 11.39 | 95.85 ± 16.48 | 108.26 ± 5.15 | 0.43 |
lungs (mg/kg) | 77.87 ± 11.39 | 72.59 ± 16.48 | 76.14 ± 5.15 | 0.68 |
heart (mg/kg) | 32.25 ± 4.81a | 44.28 ± 1.70b | 48.57 ± 2.91b | 0.012 |
gallbladder (mg/kg) | 37.09 ± 4.03 | 45.61 ± 2.73 | 40.80 ± 9.23 | 0.43 |
duodenum (mg/kg) | 32.25 ± 1.27 | 24.81 ± 6.42 | 39.45 ± 5.52 | 0.13 |
empty stomach (mg/kg) | 22.17 ± 2.99 | 19.38 ± 1.18 | 27.05 ± 6.08 | 0.42 |
ileum (mg/kg) | 19.49 ± 4.23 | 25.26 ± 8.39 | 31.56 ± 5.28 | 0.42 |
appendix (mg/kg) | 44.54 ± 9.04 | 35.89 ± 6.67 | 44.29 ± 6.53 | 0.6 |
Samples | Total Reads | Clean Reads | Clean Bases | GC Content | % ≥ Q30 | Mapped Reads |
---|---|---|---|---|---|---|
DGZA1 | 79,413,318 | 39,706,659 | 11,869,181,074 | 50.23% | 93.59% | 76,416,860 (96.23%) |
DGZA2 | 43,121,842 | 21,560,921 | 6,454,203,722 | 49.27% | 93.94% | 41,822,247 (96.99%) |
DGZA3 | 40,057,702 | 20,028,851 | 5,995,758,474 | 49.01% | 93.44% | 38,763,058 (96.77%) |
DGZB2 | 42,296,658 | 21,148,329 | 6,331,325,796 | 49.27% | 94.67% | 41,102,259 (97.18%) |
DGZB4 | 39,758,812 | 19,879,406 | 5,937,886,126 | 50.75% | 93.20% | 38,221,228 (96.13%) |
DGZB5 | 73,804,472 | 36,902,236 | 11,043,034,970 | 49.66% | 93.69% | 70,894,004 (96.06%) |
DGZC1 | 40,283,306 | 20,141,653 | 6,029,912,910 | 50.16% | 94.08% | 38,966,953 (96.73%) |
DGZC2 | 38,679,518 | 19,339,759 | 5,787,524,674 | 49.62% | 94.28% | 37,324,752 (96.50%) |
DGZC7 | 43,304,726 | 21,652,363 | 6,476,755,962 | 50.20% | 94.73% | 41,881,315 (96.71%) |
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Qian, H.; Wang, P.; Li, T.; Zhang, C.; Li, J.; Wang, Q.; Ren, H.; Jin, F.; Huang, J.; Yao, J.; et al. Joint Transcriptomic Analysis of the Effect of Iron Concentration on Piglet Liver and Functional Validation of Iron Regulatory Genes. Curr. Issues Mol. Biol. 2025, 47, 843. https://doi.org/10.3390/cimb47100843
Qian H, Wang P, Li T, Zhang C, Li J, Wang Q, Ren H, Jin F, Huang J, Yao J, et al. Joint Transcriptomic Analysis of the Effect of Iron Concentration on Piglet Liver and Functional Validation of Iron Regulatory Genes. Current Issues in Molecular Biology. 2025; 47(10):843. https://doi.org/10.3390/cimb47100843
Chicago/Turabian StyleQian, Haiming, Ping Wang, Tengchuan Li, Chunyong Zhang, Jintao Li, Qingliang Wang, Haiyang Ren, Fanyu Jin, Jie Huang, Jun Yao, and et al. 2025. "Joint Transcriptomic Analysis of the Effect of Iron Concentration on Piglet Liver and Functional Validation of Iron Regulatory Genes" Current Issues in Molecular Biology 47, no. 10: 843. https://doi.org/10.3390/cimb47100843
APA StyleQian, H., Wang, P., Li, T., Zhang, C., Li, J., Wang, Q., Ren, H., Jin, F., Huang, J., Yao, J., Pan, H., Guo, R., & An, Q. (2025). Joint Transcriptomic Analysis of the Effect of Iron Concentration on Piglet Liver and Functional Validation of Iron Regulatory Genes. Current Issues in Molecular Biology, 47(10), 843. https://doi.org/10.3390/cimb47100843